Pollen preferred promoters and methods of use

ABSTRACT

Compositions and methods for regulating expression of heterologous nucleotide sequences in a plant are provided. Compositions include nucleotide sequences encompasses a strong pollen preferred promoter which drives strong, specific expression of gene products in pollen. Also provided is a method for expressing a heterologous nucleotide sequence in a plant using a promoter sequence disclosed herein.

FIELD OF THE DISCLOSURE

The present disclosure relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE DISCLOSURE

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of operably linked regulatory elements that arefunctional within the plant host. Choice of the promoter sequence willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where expression in specific tissues or organs isdesired, tissue-preferred promoters may be used. Where gene expressionin response to a stimulus is desired, inducible promoters are theregulatory element of choice. In contrast, where continuous expressionis desired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in the expressionconstructs of transformation vectors to bring about varying levels ofexpression of heterologous nucleotide sequences in a transgenic plant.

Frequently it is desirable to express a DNA sequence in particulartissues or organs of a plant. For example, increased resistance of aplant to infection by soil- and air-borne pathogens might beaccomplished by genetic manipulation of the plant's genome to comprise atissue-preferred promoter operably linked to a heterologouspathogen-resistance gene such that pathogen-resistance proteins areproduced in the desired plant tissue. Alternatively, it might bedesirable to inhibit expression of a native DNA sequence within aplant's tissues to achieve a desired phenotype. In this case, suchinhibition might be accomplished with transformation of the plant tocomprise a tissue-preferred promoter operably linked to an antisensenucleotide sequence, such that expression of the antisense sequenceproduces an RNA transcript that interferes with translation of the mRNAof the native DNA sequence.

Additionally, it may be desirable to express a DNA sequence in planttissues that are in a particular growth or developmental phase such as,for example, cell division or elongation. Such a DNA sequence may beused to promote or inhibit plant growth processes, thereby affecting thegrowth rate or architecture of the plant.

Isolation and characterization of pollen preferred promoters,particularly promoters that can serve as regulatory elements forexpression of isolated nucleotide sequences of interest, are needed forimpacting various traits in plants and for use with scorable markers.

BRIEF SUMMARY OF THE DISCLOSURE

Compositions and methods for regulating gene expression in a plant areprovided. Compositions comprise novel nucleotide sequences for apromoter active in pollen tissues before, during, and/or after pollengermination. Certain embodiments of the disclosure comprise thenucleotide sequence set forth in SEQ ID NO: 53, 54, 55 and 56 andfunctional fragments thereof which drive pollen preferred-preferredexpression of an operably-linked nucleotide sequence. Embodiments of thedisclosure also include DNA constructs comprising a promoter operablylinked to a heterologous nucleotide sequence of interest, wherein saidpromoter is capable of driving expression of said nucleotide sequence ina plant cell and said promoter comprises one of the nucleotide sequencesdisclosed herein. Embodiments of the disclosure further provideexpression vectors, and plants or plant cells having stably incorporatedinto their genomes a DNA construct as is described above. Additionally,compositions include transgenic seed of such plants

Further embodiments comprise a means for selectively expressing anucleotide sequence in a plant, comprising transforming a plant cellwith a DNA construct, and regenerating a transformed plant from saidplant cell, said DNA construct comprising a promoter of the disclosureand a heterologous nucleotide sequence operably linked to said promoter,wherein said promoter initiates pollen preferred-preferred transcriptionof said nucleotide sequence in the regenerated plant. In this manner,the promoter sequences are useful for controlling the expression ofoperably linked coding sequences in a tissue-preferred manner.

Downstream from the transcriptional initiation region of the promoterwill be a sequence of interest that will provide for modification of thephenotype of the plant. Such modification includes modulating theproduction of an endogenous product as to amount, relative distribution,or the like, or production of an exogenous expression product, toprovide for a novel or modulated function or product in the plant. Forexample, a heterologous nucleotide sequence that encodes a gene productthat confers resistance or tolerance to herbicide, salt, cold, drought,pathogen, nematodes or insects is encompassed.

In a further embodiment, a method for modulating expression of a gene ina stably transformed plant is provided, comprising the steps of (a)transforming a plant cell with a DNA construct comprising the promoterof the disclosure operably linked to at least one nucleotide sequence;(b) growing the plant cell under plant growing conditions and (c)regenerating a stably transformed plant from the plant cell whereinexpression of the linked nucleotide sequence alters the phenotype of theplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 At-LAT52 LP1 Pro: AC-GFP1 signal found to be very bright inpollen, note shed pollen on carpel wall.

FIG. 2A and FIG. 2B At-LAT52 LP1 Pro: AC-GFP1—Anthers. GFP pollensegregates for signal as expected. Note auto-fluorescence of anther(FIG. 2A), but not pollen in the negative control (FIG. 2B).

FIG. 3A and FIG. 3B At-LAT52 LP1 Pro: AC-GFP1—Pollen Grains. GFP pollensegregates for signal as expected.

FIG. 4A and FIG. 4B AT-LAT52LP2 PRO:AC-GFP1—GFP pollen segregates forsignal as expected.

FIG. 5A and FIG. 5B AT-PPG1 PRO:AC-GFP1—GFP pollen demonstratesfluorescence and segregates for signal as expected.

FIG. 6A, FIG. 6B, and FIG. 6C AT-LAT52 LP2 PRO:BARNASE-BARNASEconstructs tended to have plieotropic effects on the flowers. (FIG. 6A)short or missing petals, (FIG. 6B) disfigured outer floral organs and(FIG. 6C) disfigured outer floral organs.

FIG. 7A and FIG. 7B AT-PPG1 PRO:BARNASE appeared more normal thanAT-LAT52 LP2:BARNASE. (FIG. 7A) non-transformed control (FIG. 7B) istransformed with BARNASE construct.

FIG. 8A, FIG. 8B, and FIG. 8C AT-LAT52LP1 PRO:BARNASE (FIG. 8A) Stuntedpetals, some floral plieotropism. In seed—(FIG. 8B) white light (FIG.8C) fluorescent YFP filtered—the seed does segregate 1:1 as expected.

FIG. 9A and FIG. 9B AT-LAT52LP2 PRO:ADP Ribosylase In seed—(FIG. 9A)white light (FIG. 9B) fluorescent YFP filtered—the seed does segregate1:1 as expected. FIG. 10A and FIG. 10B AT-LAT52LP1 PRO:DMETH Inseed—(FIG. 10A) white light (FIG. 10B) fluorescent YFP filtered—the seeddoes segregate 1:1 as expected.

FIG. 11A and FIG. 11B AT-LAT52LP2 PRO:DMETH In seed—(FIG. 11A) whitelight (FIG. 11B) fluorescent YFP filtered—the seed does segregate 1:1 asexpected.

FIG. 12A, FIG. 12B, and FIG. 12C AT-PPG2 PRO:GUS (FIG. 12A) pollenpreferred staining, (FIG. 12B) GUS staining shows expression signalstill found in the pollen tubes and (FIG. 12C) showing a magnified viewof an intact anther with expression confined to pollen grains.

FIG. 13 is a fluorescent image of a fertilized Arabidopsis embryo sacwith only remnants of the egg/zygote (red) and of the synergids (green).Mixing of the breakdown products green and red equal yellow. Centralcell appears healthy with 3-4 endosperm nuclei indicating thatfertilization did occur.

FIG. 14 is a fluorescent image of a fertilized Arabidopsis embryo sacwith a zygote (red) that is in the process of breaking down, losingintegrity and appears to be “blebbing”. The persistent synergid (green)appears to be condensing and breaking down as well. Central cell appearshealthy with several endosperm nuclei indicating that fertilization didoccur.

FIG. 15 is a fluorescent image of a fertilized Arabidopsis embryo sacshowing 7-8 endosperm nuclei in a normal developing central cell. Nosign of a zygote or embryo (red) nor any sign of a synergid (green) ispresent. The endosperm may be described as developing in the absence ofan embryo.

FIG. 16 is a fluorescent image of a fertilized Arabidopsis embryo sacwith a remnant of the zygote (red) and the persistent synergid (green),where both appear to be condensing and breaking down. Central cellappears to be unhealthy and in the early stages of breaking down as isindicated by the increased vacuolation of the central cell.

FIG. 17 is a fluorescent image of 2 unfertilized Arabidopsis embryo sacsjust prior to fertilization. The embryo sac at left has a central cell(cyan) with the 2 endosperm nuclei and 2 synergids (yellow), but islacking an egg (red). The embryo sac at right has a central cell (cyan)with the single primary endosperm nucleus, but is lacking the synergids(yellow) and the egg (red).

FIG. 18A and FIG. 18B is a fluorescent and differential interferencecontrast (DIC) fluorescent overlay image of a fertilized Arabidopsisembryo sac. The central cell (cyan) has the single endosperm nucleus and1 synergid (yellow), but is lacking an egg (arrow).

FIG. 19 is a fluorescent image of a fertilized Arabidopsis embryo sacwith 4 endosperm nuclei in a normal developing central cell. Only a veryweak red fluorescent signal (arrow) indicative of a remnant of theembryo or zygote is present. The persistent synergid (green) is breakingdown. The endosperm is developing in the absence of an embryo.

FIG. 20 is a fluorescent image of 2 Arabidopsis embryo sacs with welldeveloped endosperm. The embryo sac at left has numerous endospermnuclei in its central cell (cyan) and at its micropylar end (arrow) is aremnant of the embryo or zygote (red). Under normal conditions thisembryo should be much more fully developed, at the heart-shaped stage.The smaller embryo sac at right has numerous endosperm nuclei (cyan) butis lacking an embryo (arrow). Synergids would have been lost by thislate stage.

DETAILED DESCRIPTION

The disclosure relates to compositions and methods drawn to plantpromoters and methods of their use. The compositions comprise nucleotidesequences for a pollen preferred promoters. The compositions furthercomprise DNA constructs comprising a nucleotide sequence for thepromoter region operably linked to a heterologous nucleotide sequence ofinterest. In particular, the present disclosure provides for isolatednucleic acid molecules comprising the nucleotide sequence set forth inSEQ ID NOS: 53, 54, 55 and 56, and fragments, variants and complementsthereof.

The promoter sequences of the present disclosure include nucleotideconstructs that allow initiation of transcription in a plant. Inspecific embodiments, the promoter sequence allows initiation oftranscription in a tissue-preferred manner, more particularly in apollen preferred manner. Such constructs of the disclosure compriseregulated transcription initiation regions associated with plantdevelopmental regulation. Thus, the compositions of the presentdisclosure include DNA constructs comprising a nucleotide sequence ofinterest operably linked to a plant promoter, particularly a pollenpreferred promoter sequence, more particularly an Arabidopsis pollenpromoter sequence. A sequence comprising the Arabidopsis pollen promoterregion is set forth in SEQ ID NOS: 53, 54, 55 and 56.

TABLE 1 POLYNUCLEOTIDE/ POLYPEPTIDE SEQ ID. NAME DESCRIPTION (PN/PP) SEQID NO: 1 AT-NUC1 PRO OVULE TISSUE- PN (AT4G21620) PREFERRED PROMOTER SEQID NO: 2 ALT-AT-NUC1 OVULE TISSUE- PN PRO PREFERRED (AT4G21620) PROMOTERSEQ ID NO: 3 AT-CYP86C1 OVULE TISSUE- PN (AT1G24540) PREFERRED PROMOTERSEQ ID NO: 4 ALT-AT- OVULE TISSUE- PN CYP86C1 PREFERRED PROMOTER SEQ IDNO: 5 AT-PPM1 PRO OVULE TISSUE- PN AT5G49180 PREFERRED PROMOTER SEQ IDNO: 6 AT-EXT PRO OVULE TISSUE- PN AT3G48580 PREFERRED PROMOTER SEQ IDNO: 7 AT-GILT1 PRO OVULE TISSUE- PN AT4G12890 PREFERRED PROMOTER SEQ IDNO: 8 AT-TT2 PRO OVULE TISSUE- PN AT5G35550 PREFERRED PROMOTER SEQ IDNO: 9 AT-SVL3 PRO OVULE TISSUE- PN PREFERRED PROMOTER SEQ ID NO: 10AT-DD45 PRO EGG CELL-PREFERRED PN PROMOTER SEQ ID NO: 11 ATRKD1 CDNA OFRKD PN FULL LENGTH POLYPEPTIDE CDNA SEQ ID NO: 12 ATRKD1 RKD POLYPEPTIDEPP AMINO ACID NM_101737.1 SEQ ID NO: 13 ATRKD2 CDNA OF RKD PN(AT1G74480) POLYPEPTIDE FULL LENGTH CDNA NM_106108 SEQ ID NO: 14 ATRKD2RKD POLYPEPTIDE PP (AT1G74480) AMINO ACID SEQ ID NO: 15 ATRKD3 CDNA OFRKD PN (AT5G66990) POLYPEPTIDE FULL LENGTH CDNA NM_126099 SEQ ID NO: 16ATRKD3 RKD POLYPEPTIDE PP (AT5G66990) AMINO ACID NP_201500.1 SEQ ID NO:17 ATRKD4 CDNA OF RKD PN (AT5G53040) POLYPEPTIDE FULL LENGTH CDNA SEQ IDNO: 18 ATRKD4 RKD POLYPEPTIDE PP (AT5G53040) AMINO ACID NP_200116.1 SEQID NO: 19 EASE PRO EGG CELL-PREFERRED PN PROMOTER SEQ ID NO: 20 AT-DD2PRO EGG CELL-PREFERRED PN PROMOTER SEQ ID NO: 21 AT-RKD1 PRO EGGCELL-PREFERRED PN SEQ ID NO: 22 AT-RKD2 PRO EGG CELL-PREFERRED PN SEQ IDNO: 23 BA-BARNASE- DNA ENCODING PN INT CYTOTOXIC POLYPEPTIDE SEQ ID NO:24 DAM DNA ENCODING PN METHYLASE CYTOTOXIC POLYPEPTIDE SEQ ID NO: 25DMETH N-TERM OLIGONUCLEOTIDE PN SEQ ID NO: 26 INTE-N OLIGONUCLEOTIDE PNSEQ ID NO: 27 INTE-C OLIGONUCLEOTIDE PN SEQ ID NO: 28 DMETH C-TERMOLIGONUCLEOTIDE PN SEQ ID NO: 29 ADP DNA ENCODING PN RIBOSYLASECTYOTOXIC POLYPEPTIDE SEQ ID NO: 30 FEM2 EMBRYO SAC- PN PREFERREDPROMOTER SEQ ID NO: 31 ATRKD5 CDNA OF RKD PN AT4G35590; DNA; POLYPEPTIDEARABIDOPSIS THALIANA SEQ ID NO: 32 AT- RKD POLYPEPTIDE PP RKD5; PRT;ARABIDOPSIS THALIANA SEQ ID NO: 33 AT1G24540 OVULE TISSUE- PN AT-CP450-1PRO PREFERRED PROMOTER SEQ ID NO: 34 ZMDD45PRO; PROMOTER PN DNA; ZEAMAYS SEQ ID NO: 35 PCO6594805PRIMELONG; OLIGONUCLEOTIDE PN DNA; ZEA MAYSSEQ ID NO: 36 PCO6594803PRIMELONG; OLIGONUCLEOTIDE PN DNA; ZEA MAYS SEQID NO: 37 ZSGREEN5PRIME; OLIGONUCLEOTIDE PN DNA; ZOANTHUS SP SEQ ID NO:38 ZSGREEN3PRIME; OLIGONUCLEOTIDE PN DNA; ZOANTHUS SP SEQ ID NO: 39CYAN1 5PRIME; OLIGONUCLEOTIDE PN DNA; ANEMONIA MAJANO SEQ ID NO: 40CYAN1 3PRIME; OLIGONUCLEOTIDE PN DNA; ANEMONIA MAJANO SEQ ID NO: 41AT-DD1 PRO; PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 42 AT-DD31PRO; PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 43 AT-DD65 PRO;PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 44 SORGHUM PROMOTER -OVULE PN BICOLOR OVULE SPECIFIC PROMOTER 1 (SB10G008120.1) SEQ ID NO: 45PROMOTER PROMOTER - OVULE PN RICE OVULE CANDIDATE 1 (OS02G-51090) SEQ IDNO: 46 AT-RKD2 PRO PROMOTER WITH PN (AT1G74480) PROPOSED TETOPSITES.OPTION 1 SEQ ID NO: 47 AT-RKD2 PRO PROMOTER WITH PN (AT1G74480) PROPOSEDTETOPSITES. OPTION 2 SEQ ID NO: 48 AT-RKD2 PRO PROMOTER WITH PN(AT1G74480) PROPOSED TETOPSITES. OPTION 3 SEQ ID NO: 49 BA-BASTAR;CYTOTOXIC COGNATE PN DNA; BACILLUS REPRESSOR AMYLOLIQUEFACIENS SEQ IDNO: 50 AT-RKD3 PRO; PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 51AT-RKD4 PRO; PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 52 AT-RKD5PRO; PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 53 AT-LAT52LP1PROMOTER PN PRO; DNA; ARABIDOPSIS THALIANA SEQ ID NO: 54 AT-LAT52LP2PROMOTER PN PRO; DNA; ARABIDOPSIS THALIANA SEQ ID NO: 55 AT-PPG1 PRO;PROMOTER PN DNA; ARABIDOPSIS THALIANA SEQ ID NO: 56 AT-PPG2 PRO;PROMOTER PN DNA; ARABIDOPSIS THALIANA

Compositions of the disclosure include the nucleotide sequences for thenative promoter and fragments and variants thereof. The promotersequences of the disclosure are useful for expressing sequences. Inspecific embodiments, the promoter sequences of the disclosure areuseful for expressing sequences of interest particularly in a pollenpreferred manner. The nucleotide sequences of the disclosure also finduse in the construction of expression vectors for subsequent expressionof a heterologous nucleotide sequence in a plant of interest or asprobes for the isolation of other pollen preferred promoters. Inparticular, the present disclosure provides for isolated DNA constructscomprising the promoter nucleotide sequence set forth in SEQ ID NO: 53,54, 55 and 56 operably linked to a nucleotide sequence of interest

The disclosure encompasses isolated or substantially purified nucleicacid compositions. An “isolated” or “purified” nucleic acid molecule orbiologically active portion thereof is substantially free of othercellular material or culture medium when produced by recombinanttechniques or substantially free of chemical precursors or otherchemicals when chemically synthesized. An “isolated” nucleic acid issubstantially free of sequences (including protein encoding sequences)that naturally flank the nucleic acid (i.e., sequences located at the 5′and 3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. The promoter sequences of thedisclosure may be isolated from the 5′ untranslated region flankingtheir respective transcription initiation sites.

Fragments and variants of the disclosed promoter nucleotide sequencesare also encompassed by the present disclosure. In particular, fragmentsand variants of the promoter sequence of SEQ ID NOS: 53-56 may be usedin the DNA constructs of the disclosure. As used herein, the term“fragment” refers to a portion of the nucleic acid sequence. Fragmentsof a promoter sequence may retain the biological activity of initiatingtranscription, more particularly driving transcription in a pollenpreferred manner. Alternatively, fragments of a nucleotide sequence thatare useful as hybridization probes may not necessarily retain biologicalactivity. Fragments of a nucleotide sequence for the promoter region mayrange from at least about 20 nucleotides, about 50 nucleotides, about100 nucleotides and up to the full length of SEQ ID NOS: 53-56.

A biologically active portion of a promoter can be prepared by isolatinga portion of the promoter sequence of the disclosure, and assessing thepromoter activity of the portion. Nucleic acid molecules that arefragments of a promoter nucleotide sequence comprise at least about 16,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700or 800 nucleotides or up to the number of nucleotides present in afull-length promoter sequence disclosed herein.

As used herein, the term “variants” is intended to mean sequences havingsubstantial similarity with a promoter sequence disclosed herein. Avariant comprises a deletion and/or addition of one or more nucleotidesat one or more internal sites within the native polynucleotide and/or asubstitution of one or more nucleotides at one or more sites in thenative polynucleotide. As used herein, a “native” nucleotide sequencecomprises a naturally occurring nucleotide sequence. For nucleotidesequences, naturally occurring variants can be identified with the useof well-known molecular biology techniques, such as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedherein.

Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis. Generally, variants of a particularnucleotide sequence of the embodiments will have at least 40%, 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, to 95%, 96%, 97%, 98%,99% or more sequence identity to that particular nucleotide sequence asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. Biologically active variants are alsoencompassed by the embodiments. Biologically active variants include,for example, the native promoter sequences of the embodiments having oneor more nucleotide substitutions, deletions or insertions. Promoteractivity may be measured by using techniques such as Northern blotanalysis, reporter activity measurements taken from transcriptionalfusions, and the like. See, for example, Sambrook, et al., (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), hereinafter “Sambrook,”herein incorporated by reference in its entirety. Alternatively, levelsof a reporter gene such as green fluorescent protein (GFP) or yellowfluorescent protein (YFP) or the like produced under the control of apromoter fragment or variant can be measured. See, for example, Matz, etal., (1999) Nature Biotechnology 17:969-973; U.S. Pat. No. 6,072,050,herein incorporated by reference in its entirety; Nagai, et al., (2002)Nature Biotechnology 20(1):87-90. Variant nucleotide sequences alsoencompass sequences derived from a mutagenic and recombinogenicprocedure such as DNA shuffling. With such a procedure, one or moredifferent nucleotide sequences for the promoter can be manipulated tocreate a new promoter. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer, (1994) Nature 370:389 391; Crameri, et al., (1997) NatureBiotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347;Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri,et al., (1998) Nature 391:288-291 and U.S. Pat. Nos. 5,605,793 and5,837,458, herein incorporated by reference in their entirety.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Kunkel, (1985) Proc. Natl. Acad.Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in Enzymol.154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, New York)and the references cited therein, herein incorporated by reference intheir entirety.

The nucleotide sequences of the disclosure can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other dicots. In this manner, methods such as PCR,hybridization and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire sequences setforth herein or to fragments thereof are encompassed by the presentdisclosure.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in, Sambrook, supra. See also, Innis, et al., eds. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York), herein incorporated by reference in theirentirety. Known methods of PCR include, but are not limited to, methodsusing paired primers, nested primers, single specific primers,degenerate primers, gene-specific primers, vector-specific primers,partially-mismatched primers and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides and may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the promoter sequences ofthe disclosure. Methods for preparation of probes for hybridization andfor construction of genomic libraries are generally known in the art andare disclosed in Sambrook, supra.

For example, the entire promoter sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding dicot pollen promoter sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among promotersequences and are generally at least about 10 nucleotides in length orat least about 20 nucleotides in length. Such probes may be used toamplify corresponding promoter sequences from a chosen plant by PCR.This technique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies, see, for example, Sambrook, supra).

Hybridization of such sequences may be carried out under stringentconditions. The terms “stringent conditions” or “stringent hybridizationconditions” are intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.and a wash in 1 times to 2 times SSC (20 times SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1%SDS at 37° C. and a wash in 0.5 times to 1 times SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a final wash in 0.1 times SSCat 60 to 65° C. for a duration of at least 30 minutes. Duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours. The duration of the wash time will be at least a lengthof time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the thermal melting point (T_(m))can be approximated from the equation of Meinkoth and Wahl, (1984) Anal.Biochem 138:267 284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching, thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with 90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3 or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9 or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York) and Ausubel, et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York), herein incorporated byreference in their entirety. See also, Sambrook.

Thus, isolated sequences that have pollen preferred promoter activityand which hybridize under stringent conditions to the promoter sequencesdisclosed herein or to fragments thereof, are encompassed by the presentdisclosure.

In general, sequences that have promoter activity and hybridize to thepromoter sequences disclosed herein will be at least 40% to 50%homologous, about 60%, 70%, 80%, 85%, 90%, 95% to 98% homologous or morewith the disclosed sequences. That is, the sequence similarity ofsequences may range, sharing at least about 40% to 50%, about 60% to70%, and about 80%, 85%, 90%, 95% to 98% sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity” and (e) “substantial identity”.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100 or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence, a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, (1988) CABIOS 4:11-17; the algorithm of Smith, etal., (1981) Adv. Appl. Math. 2:482; the algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443-453; the algorithm of Pearson andLipman, (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm ofKarlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 872:264, modifiedas in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA90:5873-5877, herein incorporated by reference in their entirety.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA andTFASTA in the GCG Wisconsin Genetics Software Package®, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins, et al.,(1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153;Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al.,(1992) CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-331, herein incorporated by reference in their entirety. TheALIGN program is based on the algorithm of Myers and Miller, (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul, et al., (1990) J. Mol.Biol. 215:403, herein incorporated by reference in its entirety, arebased on the algorithm of Karlin and Altschul, (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,word length=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding a protein of the disclosure. BLAST proteinsearches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein orpolypeptide of the disclosure. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul, et al., (1997) Nucleic Acids Res. 25:3389, hereinincorporated by reference in its entirety. Alternatively, PSI-BLAST (inBLAST 2.0) can be used to perform an iterated search that detectsdistant relationships between molecules. See, Altschul, et al., (1997)supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the defaultparameters of the respective programs (e.g., BLASTN for nucleotidesequences, BLASTX for proteins) can be used. See, the web site for theNational Center for Biotechnology Information on the World Wide Web atncbi.nlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. As usedherein, “equivalent program” is any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

The GAP program uses the algorithm of Needleman and Wunsch, supra, tofind the alignment of two complete sequences that maximizes the numberof matches and minimizes the number of gaps. GAP considers all possiblealignments and gap positions and creates the alignment with the largestnumber of matched bases and the fewest gaps. It allows for the provisionof a gap creation penalty and a gap extension penalty in units ofmatched bases. GAP must make a profit of gap creation penalty number ofmatches for each gap it inserts. If a gap extension penalty greater thanzero is chosen, GAP must, in addition, make a profit for each gapinserted of the length of the gap times the gap extension penalty.Default gap creation penalty values and gap extension penalty values inVersion 10 of the GCG Wisconsin Genetics Software Package® for proteinsequences are 8 and 2, respectively. For nucleotide sequences thedefault gap creation penalty is 50 while the default gap extensionpenalty is 3. The gap creation and gap extension penalties can beexpressed as an integer selected from the group of integers consistingof from 0 to 200. Thus, for example, the gap creation and gap extensionpenalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage® is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915, herein incorporated by reference in itsentirety).

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of one and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and one. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, optimally at least 80%, more optimally at least 90% and mostoptimally at least 95%, compared to a reference sequence using analignment program using standard parameters. One of skill in the artwill recognize that these values can be appropriately adjusted todetermine corresponding identity of proteins encoded by two nucleotidesequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. Substantial identityof amino acid sequences for these purposes normally means sequenceidentity of at least 60%, 70%, 80%, 90% and at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The promoter sequence disclosed herein, as well as variants andfragments thereof, are useful for genetic engineering of plants, e.g.,for the production of a transformed or transgenic plant, to express aphenotype of interest. As used herein, the terms “transformed plant” and“transgenic plant” refer to a plant that comprises within its genome aheterologous polynucleotide. Generally, the heterologous polynucleotideis stably integrated within the genome of a transgenic or transformedplant such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant DNA construct. It is to beunderstood that as used herein the term “transgenic” includes any cell,cell line, callus, tissue, plant part or plant the genotype of which hasbeen altered by the presence of heterologous nucleic acid includingthose transgenics initially so altered as well as those created bysexual crosses or asexual propagation from the initial transgenic.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid expression cassettethat comprises a transgene of interest, the regeneration of a populationof plants resulting from the insertion of the transgene into the genomeof the plant and selection of a particular plant characterized byinsertion into a particular genome location. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic makeup of a plant. The term “event” alsorefers to progeny produced by a sexual cross between the transformantand another plant wherein the progeny include the heterologous DNA.

As used herein, the term plant includes whole plants, plant organs(e.g., leaves, stems, roots, etc.), plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps and plant cells that are intact in plants or partsof plants such as embryos, pollen, developing microspores, seeds,leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks,roots, root tips, anthers and the like. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants and mutants of theregenerated plants are also included within the scope of the disclosure,provided that these parts comprise the introduced polynucleotides.

The present disclosure may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species include corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis) and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima) and chrysanthemum.

Conifers that may be employed in practicing the present disclosureinclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinusponderosa), lodgepole pine(Pinus contorta) and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea) and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent disclosure are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Heterologous coding sequences expressed by a promoter of the disclosuremay be used for varying the phenotype of a plant. Various changes inphenotype are of interest including modifying expression of a gene in aplant, altering a plant's pathogen or insect defense mechanism, changinga plant's reproductive capacities, preventing paternal transgenetransmission, increasing a plant's tolerance to herbicides, alteringplant development to respond to environmental stress, modulating theplant's response to salt, temperature (hot and cold), drought and thelike. These results can be achieved by the expression of a heterologousnucleotide sequence of interest comprising an appropriate gene product.In specific embodiments, the heterologous nucleotide sequence ofinterest is an endogenous plant sequence whose expression level isincreased in the plant or plant part. Results can be achieved byproviding for altered expression of one or more endogenous geneproducts, particularly hormones, receptors, signaling molecules,enzymes, transporters or cofactors or by affecting nutrient uptake inthe plant. Tissue-preferred expression as provided by the promoter cantarget the alteration in expression to plant parts and/or growth stagesof particular interest, such as developing microspores, particularly thepollen. These changes result in a change in phenotype of the transformedplant

General categories of nucleotide sequences of interest for the presentdisclosure include, for example, those genes involved in information,such as zinc fingers, those involved in communication, such as kinasesand those involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes, for example, include genes encodingimportant traits for agronomics, insect resistance, disease resistance,herbicide resistance, environmental stress resistance (altered toleranceto cold, salt, drought, etc) and grain characteristics. Still othercategories of transgenes include genes for inducing expression ofexogenous products such as enzymes, cofactors, and hormones from plantsand other eukaryotes as well as prokaryotic organisms. It is recognizedthat any gene of interest can be operably linked to the promoter of thedisclosure and expressed in the plant.

Agronomically important traits that affect quality of grain, such aslevels and types of oils, saturated and unsaturated, quality andquantity of essential amino acids, levels of cellulose, starch andprotein content can be genetically altered using the methods of theembodiments. Modifications to grain traits include, but are not limitedto, increasing content of oleic acid, saturated and unsaturated oils,increasing levels of lysine and sulfur, providing essential amino acids,and modifying starch. Hordothionin protein modifications in corn aredescribed in U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and5,703,049; herein incorporated by reference in their entirety. Anotherexample is lysine and/or sulfur rich seed protein encoded by the soybean2S albumin described in U.S. Pat. No. 5,850,016, filed Mar. 20, 1996 andthe chymotrypsin inhibitor from barley, Williamson, et al., (1987) Eur.J. Biochem 165:99-106, the disclosures of which are herein incorporatedby reference in their entirety.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European corn borer and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes, U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881 and Geiser, et al., (1986) Gene 48:109, the disclosures ofwhich are herein incorporated by reference in their entirety. Genesencoding disease resistance traits include, for example, detoxificationgenes, such as those which detoxify fumonisin (U.S. Pat. No. 5,792,931);avirulence (avr) and disease resistance (R) genes (Jones, et al., (1994)Science 266:789; Martin, et al., (1993) Science 262:1432; and Mindrinos,et al., (1994) Cell 78:1089), herein incorporated by reference in theirentirety.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), genescoding for resistance to glyphosate (e.g., the EPSPS gene and the GATgene; see, for example, US Patent Application Publication Number2004/0082770 and WO 2003/092360, herein incorporated by reference intheir entirety) or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotics kanamycin and geneticin and the ALS-genemutants encode resistance to the herbicide chlorsulfuron.

Glyphosate resistance is imparted by mutant 5-enolpyruvl-3-phosphikimatesynthase (EPSP) and aroA genes. See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; Re. 36,449; RE 37,287 E and 5,491,288 and internationalpublications WO 1997/04103; WO 1997/04114; WO 2000/66746; WO 2001/66704;WO 2000/66747 and WO 2000/66748, which are incorporated herein byreference in their entirety. Glyphosate resistance is also imparted toplants that express a gene that encodes a glyphosate oxido-reductaseenzyme as described more fully in U.S. Pat. Nos. 5,776,760 and5,463,175, which are incorporated herein by reference in their entirety.In addition glyphosate resistance can be imparted to plants by the overexpression of genes encoding glyphosate N-acetyltransferase. See, forexample, U.S. patent application Ser. Nos. 11/405,845 and 10/427,692,herein incorporated by reference in their entirety.

Sterility genes can also be encoded in a DNA construct and provide analternative to physical detasseling. Examples of genes used in such waysinclude male tissue-preferred genes and genes with male sterilityphenotypes such as QM, described in U.S. Pat. No. 5,583,210, hereinincorporated by reference in its entirety. Other genes include kinasesand those encoding compounds toxic to either male or female gametophyticdevelopment.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321, herein incorporated by reference in itsentirety. Genes such as δ-Ketothiolase, PHBase (polyhydroxybutyratesynthase), and acetoacetyl-CoA reductase (see, Schubert, et al., (1988)J. Bacteriol. 170:5837-5847, herein incorporated by reference in itsentirety) facilitate expression of polyhydroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones and the like.

Examples of other applicable genes and their associated phenotypeinclude the gene which encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that promote yield improvement and genes thatprovide for resistance to stress, such as cold, dehydration resultingfrom drought, heat and salinity, toxic metal or trace elements or thelike.

By way of illustration, without intending to be limiting, the followingis a list of other examples of the types of genes which can be used inconnection with the regulatory sequences of the disclosure.

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones, et aL, (1994) Science 266:789(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin, et al., (1993) Science 262:1432 (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos,et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene for resistance toPseudomonas syringae); McDowell and Woffenden, (2003) Trends Biotechnol.21(4):178-83 and Toyoda, et al., (2002) Transgenic Res. 11(6):567-82,herein incorporated by reference in their entirety. A plant resistant toa disease is one that is more resistant to a pathogen as compared to thewild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,(1986) Gene 48:109, who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Numbers40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581; WO 1997/40162and U.S. application Ser. Nos. 10/032,717; 10/414,637 and 10/606,320,herein incorporated by reference in their entirety.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., (1990) Nature 344:458, of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone,herein incorporated by reference in its entirety.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, (1994) J. Biol. Chem. 269:9 (expression cloning yields DNA codingfor insect diuretic hormone receptor); Pratt, et al., (1989) Biochem.Biophys. Res. Comm. 163:1243 (an allostatin is identified in Diplopterapuntata); Chattopadhyay, et al., (2004) Critical Reviews in Microbiology30(1):33-54; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini andGrossi-de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al., (2001)Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon44(4):385-403, herein incorporated by reference in their entirety. Seealso, U.S. Pat. No. 5,266,317 to Tomalski, et al., who disclose genesencoding insect-specific toxins, herein incorporated by reference in itsentirety.

(E) An enzyme responsible for a hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See, PCTApplication Number WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene, herein incorporatedby reference in its entirety. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Numbers 39637 and 67152. See also, Kramer, et al.,(1993) Insect Biochem. Molec. Biol. 23:691, who teach the nucleotidesequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck,et al., (1993) Plant Molec. Biol. 21:673, who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene, U.S. patentapplication Ser. Nos. 10/389,432, 10/692,367 and U.S. Pat. No.6,563,020, herein incorporated by reference in their entirety.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., (1994) Plant Molec. Biol. 24:757, ofnucleotide sequences for mung bean calmodulin cDNA clones and Griess, etal., (1994) Plant Physiol. 104:1467, who provide the nucleotide sequenceof a maize calmodulin cDNA clone, herein incorporated by reference intheir entirety.

(H) A hydrophobic moment peptide. See, PCT Application Number WO1995/16776 and U.S. Pat. No. 5,580,852 (disclosure of peptidederivatives of Tachyplesin which inhibit fungal plant pathogens) and PCTApplication Number WO 1995/18855 and U.S. Pat. No. 5,607,914) (teachessynthetic antimicrobial peptides that confer disease resistance), hereinincorporated by reference in their entirety.

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43,of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum,herein incorporated by reference in its entirety.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy, et al., (1990) Ann. Rev.Phytopathol. 28:451, herein incorporated by reference in its entirety.Coat protein-mediated resistance has been conferred upon transformedplants against alfalfa mosaic virus, cucumber mosaic virus, tobaccostreak virus, potato virus X, potato virus Y, tobacco etch virus,tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments), herein incorporated by reference in its entirety.

(L) A virus-specific antibody. See, for example, Tavladoraki, et al.,(1993) Nature 366:469, who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack, hereinincorporated by reference in its entirety.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See, Lamb,et al., (1992) Bio/Technology 10:1436, herein incorporated by referencein its entirety. The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart, et al., (1992) Plant J. 2:367, herein incorporated by referencein its entirety.

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., (1992) Bio/Technology 10:305, hereinincorporated by reference in its entirety, have shown that transgenicplants expressing the barley ribosome-inactivating gene have anincreased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2):128-131, Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio.7(4):456-64 and Somssich, (2003) Cell 113(7):815-6, herein incorporatedby reference in their entirety.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et aL, (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. No. 09/950,933, herein incorporated by referencein their entirety.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. No. 5,792,931, herein incorporated byreference in its entirety.

(R) Cystatin and cysteine proteinase inhibitors. See, U.S. patentapplication Ser. No. 10/947,979, herein incorporated by reference in itsentirety.

(S) Defensin genes. See, WO 2003/000863 and U.S. patent application Ser.No. 10/178,213, herein incorporated by reference in their entirety.

(T) Genes conferring resistance to nematodes. See, WO 2003/033651 andUrwin, et. al., (1998) Planta 204:472-479, Williamson (1999) Curr OpinPlant Bio. 2(4):327-31, herein incorporated by reference in theirentirety.

(U) Genes such as rcg1 conferring resistance to Anthracnose stalk rot,which is caused by the fungus Colletotrichum graminiola. See, Jung, etal., (1994) Theor. Appl. Genet. 89:413-418, as well as, U.S. ProvisionalPatent Application No. 60/675,664, herein incorporated by reference intheir entirety.

(V) Cytotoxins such as ADP Ribosylase, BA-BARNASE-INT or DMETH whichwhen expressed in the pollen prevent paternal transgene transmission tothe next generation.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet.80:449, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937 and 5,378,824 and international publication WO 1996/33270,which are incorporated herein by reference in their entirety.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes) andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 toBarry, et al., also describes genes encoding EPSPS enzymes. See also,U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425;5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835;5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061;5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and 5,491,288 andinternational publications EP 1173580; WO 2001/66704; EP 1173581 and EP1173582, which are incorporated herein by reference in their entirety.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference in their entirety. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. patent application Ser. Nos.11/405,845 and 10/427,692 and PCT Application Number US01/46227, hereinincorporated by reference in their entirety. A DNA molecule encoding amutant aroA gene can be obtained under ATCC Accession Number 39256 andthe nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai, herein incorporated by reference in its entirety. EPPatent Application Number 0 333 033 to Kumada, et al., and U.S. Pat. No.4,975,374 to Goodman, et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin, herein incorporated by reference in their entirety.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in EP Patent Numbers 0 242 246 and 0 242 236 to Leemans, etal., De Greef, et al., (1989) Bio/Technology 7:61 which describe theproduction of transgenic plants that express chimeric bar genes codingfor phosphinothricin acetyl transferase activity, herein incorporated byreference in their entirety. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1 and 5,879,903, herein incorporated by referencein their entirety. Exemplary genes conferring resistance to phenoxyproprionic acids and cycloshexones, such as sethoxydim and haloxyfop,are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, etal., (1992) Theor. Appl. Genet. 83:435, herein incorporated by referencein its entirety.

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,(1991) Plant Cell 3:169, herein incorporated by reference in itsentirety, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker, herein incorporated byreference in its entirety, and DNA molecules containing these genes areavailable under ATCC Accession Numbers 53435, 67441 and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes, et al., (1992) Biochem. J. 285:173, hereinincorporated by reference in its entirety.

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori, et al., (1995)Mol Gen Genet 246:419, herein incorporated by reference in itsentirety). Other genes that confer resistance to herbicides include: agene encoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) PlantPhysiol. 106(1):17-23), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687 and genes forvarious phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619), herein incorporated by reference in their entirety.

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1and 5,767,373; and international publication number WO 2001/12825,herein incorporated by reference in their entirety.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic, Such as:

(A) Altered fatty acids, for example, by

-   -   (1) Down-regulation of stearoyl-ACP desaturase to increase        stearic acid content of the plant. See, Knultzon, et al., (1992)        Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 (Genes for        Desaturases to Alter Lipid Profiles in Corn), herein        incorporated by reference in their entirety,    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see, U.S.        Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 1993/11245,        herein incorporated by reference in their entirety),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 2001/12800, herein incorporated by reference in its        entirety,    -   (4) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes        such as Ipa1, Ipa3, hpt or hggt. For example, see, WO        2002/42424, WO 1998/22604, WO 2003/011015, U.S. Pat. No.        6,423,886, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,825,397, US        Patent Application Publication Numbers 2003/0079247,        2003/0204870, WO 2002/057439, WO 2003/011015 and Rivera-Madrid,        et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624, herein        incorporated by reference in their entirety.

(B) Altered phosphorus content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see, Van Hartingsveldt, et        al., (1993) Gene 127:87, for a disclosure of the nucleotide        sequence of an Aspergillus niger phytase gene, herein        incorporated by reference in its entirety.    -   (2) Up-regulation of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy, et        al., (1990) Maydica 35:383 and/or by altering inositol kinase        activity as in WO 2002/059324, US Patent Application Publication        Number 2003/0009011, WO 2003/027243, US Patent Application        Publication Number 2003/0079247, WO 1999/05298, U.S. Pat. No.        6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, WO        2002/059324, US Patent Application Publication Number        2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147,        herein incorporated by reference in their entirety.

(C) Altered carbohydrates effected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648, which is incorporated by reference in its entirety) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Numbers2005/0160488 and 2005/0204418; which are incorporated by reference inits entirety). See, Shiroza, et al., (1988) J. Bacteriol. 170:810(nucleotide sequence of Streptococcus mutans fructosyltransferase gene),Steinmetz, et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequenceof Bacillus subtilis levansucrase gene), Pen, et al., (1992)Bio/Technology 10:292 (production of transgenic plants that expressBacillus licheniformis alpha-amylase), Elliot, et al., (1993) PlantMolec. Biol. 21:515 (nucleotide sequences of tomato invertase genes),Søgaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directedmutagenesis of barley alpha-amylase gene) and Fisher, et al., (1993)Plant Physiol. 102:1045 (maize endosperm starch branching enzyme II), WO1999/10498 (improved digestibility and/or starch extraction throughmodification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL,C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed bymodification of starch levels (AGP)), herein incorporated by referencein their entirety. The fatty acid modification genes mentioned above mayalso be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683, USPatent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels through alteration of aphytl prenyl transferase (ppt), WO 2003/082899 through alteration of ahomogentisate geranyl geranyl transferase (hggt), herein incorporated byreference in their entirety.

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO 1998/42831 (increased lysine), U.S. Pat.No. 5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO 1996/01905 (increased threonine), WO1995/15392 (increased lysine), US Patent Application Publication Number2003/0163838, US Patent Application Publication Number 2003/0150014, USPatent Application Publication Number 2004/0068767, U.S. Pat. No.6,803,498, WO 2001/79516, and WO 2000/09706 (Ces A: cellulose synthase),U.S. Pat. No. 6,194,638 (hemicellulose), U.S. Pat. No. 6,399,859 and USPatent Application Publication Number 2004/0025203 (UDPGdH), U.S. Pat.No. 6,194,638 (RGP), herein incorporated by reference in their entirety.

4. Genes that Control Male-Sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511,herein incorporated by reference in their entirety. In addition to thesemethods, Albertsen, et al., U.S. Pat. No. 5,432,068, herein incorporatedby reference in its entirety, describe a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility; silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant and thus creating aplant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene conferring male fertility to be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 2001/29237, herein incorporated by reference in itsentirety).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957, herein incorporated by reference in their entirety).

(C) Introduction of the barnase and the barstar gene (Paul, et aL,(1992) Plant Mol. Biol. 19:611-622, herein incorporated by reference inits entirety).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference in their entirety.

5. Genes that Create a Site for Site Specific DNA Integration

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see Lyznik, et al., (2003) Plant Cell Rep 21:925-932 and WO1999/25821, which are hereby incorporated by reference in theirentirety. Other systems that may be used include the Gin recombinase ofphage Mu (Maeser, et al., 1991; Vicki Chandler, The Maize Handbook ch.118 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto, etal., 1983) and the R/RS system of the pSR1 plasmid (Araki, et al.,1992), herein incorporated by reference in their entirety.

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see, WO 2000/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. No. 5,892,009, U.S. Pat. No. 5,965,705,U.S. Pat. No. 5,929,305, U.S. Pat. No. 5,891,859, U.S. Pat. No.6,417,428, U.S. Pat. No. 6,664,446, U.S. Pat. No. 6,706,866, U.S. Pat.No. 6,717,034, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 1998/09521 and WO 1999/38977 describinggenes, including CBF genes and transcription factors effective inmitigating the negative effects of freezing, high salinity, and droughton plants, as well as conferring other positive effects on plantphenotype; US Patent Application Publication Number 2004/0148654 and WO2001/36596, where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress; WO 2000/006341, WO 2004/090143, U.S. patentapplication Ser. No. 10/817,483 and U.S. Pat. No. 6,992,237, wherecytokinin expression is modified resulting in plants with increasedstress tolerance, such as drought tolerance, and/or increased yield,herein incorporated by reference in their entirety. Also see, WO2002/02776, WO 2003/052063, JP 2002/281975, U.S. Pat. No. 6,084,153, WO2001/64898, U.S. Pat. No. 6,177,275 and U.S. Pat. No. 6,107,547(enhancement of nitrogen utilization and altered nitrogenresponsiveness), herein incorporated by reference in their entirety. Forethylene alteration, see US Patent Application Publication Number2004/0128719, US Patent Application Publication Number 2003/0166197 andWO 2000/32761, herein incorporated by reference in their entirety. Forplant transcription factors or transcriptional regulators of abioticstress, see, e.g., US Patent Application Publication Number 2004/0098764or US Patent Application Publication Number 2004/0078852, hereinincorporated by reference in their entirety.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see, e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO1999/49064(GI), WO 2000/46358 (FRI), WO 1997/29123, U.S. Pat. No. 6,794,560, U.S.Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638and WO 2004/031349 (transcription factors), herein incorporated byreference in their entirety.

The heterologous nucleotide sequence operably linked to the promoter andits related biologically active fragments or variants disclosed hereinmay be an antisense sequence for a targeted gene. The terminology“antisense DNA nucleotide sequence” is intended to mean a sequence thatis in inverse orientation to the 5′-to-3′ normal orientation of thatnucleotide sequence. When delivered into a plant cell, expression of theantisense DNA sequence prevents normal expression of the DNA nucleotidesequence for the targeted gene. The antisense nucleotide sequenceencodes an RNA transcript that is complementary to and capable ofhybridizing to the endogenous messenger RNA (mRNA) produced bytranscription of the DNA nucleotide sequence for the targeted gene. Inthis case, production of the native protein encoded by the targeted geneis inhibited to achieve a desired phenotypic response. Modifications ofthe antisense sequences may be made as long as the sequences hybridizeto and interfere with expression of the corresponding mRNA. In thismanner, antisense constructions having 70%, 80%, 85% sequence identityto the corresponding antisense sequences may be used. Furthermore,portions of the antisense nucleotides may be used to disrupt theexpression of the target gene. Generally, sequences of at least 50nucleotides, 100 nucleotides, 200 nucleotides or greater may be used.Thus, the promoter sequences disclosed herein may be operably linked toantisense DNA sequences to reduce or inhibit expression of a nativeprotein in the plant.

“RNAi” refers to a series of related techniques to reduce the expressionof genes (see, for example, U.S. Pat. No. 6,506,559, herein incorporatedby reference in its entirety). Older techniques referred to by othernames are now thought to rely on the same mechanism, but are givendifferent names in the literature. These include “antisense inhibition,”the production of antisense RNA transcripts capable of suppressing theexpression of the target protein and “co-suppression” or“sense-suppression,” which refer to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020, incorporated herein by reference in its entirety). Suchtechniques rely on the use of constructs resulting in the accumulationof double stranded RNA with one strand complementary to the target geneto be silenced. The promoters of the embodiments may be used to driveexpression of constructs that will result in RNA interference includingmicroRNAs and siRNAs.

As used herein, the terms “promoter” or “transcriptional initiationregion” mean a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter may additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter regions disclosed herein, it is within thestate of the art to isolate and identify further regulatory elements inthe 5′ untranslated region upstream from the particular promoter regionsidentified herein. Additionally, chimeric promoters may be provided.Such chimeras include portions of the promoter sequence fused tofragments and/or variants of heterologous transcriptional regulatoryregions. Thus, the promoter regions disclosed herein can compriseupstream regulatory elements such as, those responsible for tissue andtemporal expression of the coding sequence, enhancers and the like. Inthe same manner, the promoter elements, which enable expression in thedesired tissue such as reproductive tissue, can be identified, isolatedand used with other core promoters to confer pollen preferredexpression. In this aspect of the disclosure, “core promoter” isintended to mean a promoter without promoter elements.

As used herein, the term “regulatory element” also refers to a sequenceof DNA, usually, but not always, upstream (5′) to the coding sequence ofa structural gene, which includes sequences which control the expressionof the coding region by providing the recognition for RNA polymeraseand/or other factors required for transcription to start at a particularsite. An example of a regulatory element that provides for therecognition for RNA polymerase or other transcriptional factors toensure initiation at a particular site is a promoter element. A promoterelement comprises a core promoter element, responsible for theinitiation of transcription, as well as other regulatory elements thatmodify gene expression. It is to be understood that nucleotidesequences, located within introns or 3′ of the coding region sequencemay also contribute to the regulation of expression of a coding regionof interest. Examples of suitable introns include, but are not limitedto, the maize IVS6 intron, or the maize actin intron. A regulatoryelement may also include those elements located downstream (3′) to thesite of transcription initiation, or within transcribed regions, orboth. In the context of the present disclosure a post-transcriptionalregulatory element may include elements that are active followingtranscription initiation, for example translational and transcriptionalenhancers, translational and transcriptional repressors and mRNAstability determinants.

The regulatory elements or variants or fragments thereof, of the presentdisclosure may be operatively associated with heterologous regulatoryelements or promoters in order to modulate the activity of theheterologous regulatory element. Such modulation includes enhancing orrepressing transcriptional activity of the heterologous regulatoryelement, modulating post-transcriptional events, or either enhancing orrepressing transcriptional activity of the heterologous regulatoryelement and modulating post-transcriptional events. For example, one ormore regulatory elements or fragments thereof of the present disclosuremay be operatively associated with constitutive, inducible or tissuespecific promoters or fragment thereof, to modulate the activity of suchpromoters within desired tissues in plant cells.

The regulatory sequences of the present disclosure or variants orfragments thereof, when operably linked to a heterologous nucleotidesequence of interest can drive pollen preferred expression, of theheterologous nucleotide sequence in the reproductive tissue of the plantexpressing this construct. The term “pollen preferred expression,” meansthat expression of the heterologous nucleotide sequence is most abundantin the pollen cells. While some level of expression of the heterologousnucleotide sequence may occur in other plant tissue types, expressionoccurs most abundantly in the pollen cells.

A “heterologous nucleotide sequence” is a sequence that is not naturallyoccurring with the promoter sequence of the disclosure. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous or native or heterologous or foreign to the plant host.

The isolated promoter sequences of the present disclosure can bemodified to provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter region may beutilized and the ability to drive expression of the nucleotide sequenceof interest retained. It is recognized that expression levels of themRNA may be altered in different ways with deletions of portions of thepromoter sequences. The mRNA expression levels may be decreased, oralternatively, expression may be increased as a result of promoterdeletions if, for example, there is a negative regulatory element (for arepressor) that is removed during the truncation process. Generally, atleast about 20 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence.

It is recognized that to increase transcription levels, enhancers may beutilized in combination with the promoter regions of the disclosure.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element and the like. Someenhancers are also known to alter normal promoter expression patterns,for example, by causing a promoter to be expressed constitutively whenwithout the enhancer, the same promoter is expressed only in onespecific tissue or a few specific tissues.

Modifications of the isolated promoter sequences of the presentdisclosure can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, a “weak promoter” means a promoter thatdrives expression of a coding sequence at a low level. A “low level” ofexpression is intended to mean expression at levels of about 1/10,000transcripts to about 1/100,000 transcripts to about 1/500,000transcripts. Conversely, a strong promoter drives expression of a codingsequence at a high level, or at about 1/10 transcripts to about 1/100transcripts to about 1/1,000 transcripts.

It is recognized that the promoters of the disclosure may be used withtheir native coding sequences to increase or decrease expression,thereby resulting in a change in phenotype of the transformed plant. Thenucleotide sequences disclosed in the present disclosure, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant. The promoter sequences are useful in this aspect whenoperably linked with a heterologous nucleotide sequence whose expressionis to be controlled to achieve a desired phenotypic response. The term“operably linked” means that the transcription or translation of theheterologous nucleotide sequence is under the influence of the promotersequence. In this manner, the nucleotide sequences for the promoters ofthe disclosure may be provided in expression cassettes along withheterologous nucleotide sequences of interest for expression in theplant of interest, more particularly for expression in the reproductivetissue of the plant.

In one embodiment of the disclosure, expression cassettes will comprisea transcriptional initiation region comprising one of the promoternucleotide sequences of the present disclosure, or variants or fragmentsthereof, operably linked to the heterologous nucleotide sequence. Suchan expression cassette can be provided with a plurality of restrictionsites for insertion of the nucleotide sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes as well as 3′termination regions.

The expression cassette can include, in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter, orvariant or fragment thereof, of the disclosure), a translationalinitiation region, a heterologous nucleotide sequence of interest, atranslational termination region and optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived or,if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus or the promoteris not the native promoter for the operably linked polynucleotide.

While it may be preferable to express a heterologous nucleotide sequenceusing the promoters of the disclosure, the native sequences may beexpressed. Such constructs would change expression levels of the proteinin the plant or plant cell. Thus, the phenotype of the plant or plantcell is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence beingexpressed, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144;Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev.5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al.,(1990) Gene 91:151-158; Ballas, et aL, (1989) Nucleic Acids Res.17:7891-7903; and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639,herein incorporated by reference in their entirety.

The expression cassette comprising the sequences of the presentdisclosure may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) can be provided on another expression cassette.

Where appropriate, the nucleotide sequences whose expression is to beunder the control of the pollen promoter sequence of the presentdisclosure and any additional nucleotide sequence(s) may be optimizedfor increased expression in the transformed plant. That is, thesenucleotide sequences can be synthesized using plant preferred codons forimproved expression. See, for example, Campbell and Gowri, (1990) PlantPhysiol. 92:1-11, herein incorporated by reference in its entirety, fora discussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, 5,436,391 and Murray, et al., (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference in their entirety.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats and other such well-characterized sequences thatmay be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include, without limitation:picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′noncoding region) (Elroy-Stein, et al., (1989) Proc. Nat. Acad. Sci. USA86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Allison, et al., (1986) Virology 154:9-20); MDMV leader (MaizeDwarf Mosaic Virus); human immunoglobulin heavy-chain binding protein(BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling,et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)(Gallie, et al., (1989) Molecular Biology of RNA, pages 237-256) andmaize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991)Virology 81:382-385), herein incorporated by reference in theirentirety. See, also, Della-Cioppa, et al., (1987) Plant Physiology84:965-968, herein incorporated by reference in its entirety. Methodsknown to enhance mRNA stability can also be utilized, for example,introns, such as the maize Ubiquitin intron (Christensen and Quail,(1996) Transgenic Res. 5:213-218; Christensen, et al., (1992) PlantMolecular Biology 18:675-689) or the maize Adhl intron (Kyozuka, et aL,(1991) Mol. Gen. Genet. 228:40-48; Kyozuka, et al., (1990) Maydica35:353-357) and the like, herein incorporated by reference in theirentirety.

The DNA constructs of the embodiments can also include furtherenhancers, either translation or transcription enhancers, as may berequired. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence. Thetranslation control signals and initiation codons can be from a varietyof origins, both natural and synthetic. Translational initiation regionsmay be provided from the source of the transcriptional initiationregion, or from the structural gene. The sequence can also be derivedfrom the regulatory element selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. It isrecognized that to increase transcription levels enhancers may beutilized in combination with the promoter regions of the embodiments.Enhancers are known in the art and include the SV40 enhancer region, the35S enhancer element, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

Reporter genes or selectable marker genes may also be included in theexpression cassettes of the present disclosure. Examples of suitablereporter genes known in the art can be found in, for example, Jefferson,et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al.,(Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell.Biol. 7:725-737; Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al.,(1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) CurrentBiology 6:325-330, herein incorporated by reference in their entirety.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al.,(1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron, et al., (1985)Plant Mol. Biol. 5:103-108 and Zhijian, et al., (1995) Plant Science108:219-227); streptomycin (Jones, et al., (1987) Mol. Gen. Genet.210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) TransgenicRes. 5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-36); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518), herein incorporated byreference in their entirety.

Other genes that could serve utility in the recovery of transgenicevents would include, but are not limited to, examples such as GUS(beta-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP(green fluorescence protein; Chalfie, et aL, (1994) Science 263:802),luciferase (Riggs, et al., (1987) Nucleic Acids Res. 15(19):8115 andLuehrsen, et al., (1992) Methods Enzymol. 216:397-414) and the maizegenes encoding for anthocyanin production (Ludwig, et al., (1990)Science 247:449), herein incorporated by reference in their entirety.

The expression cassette comprising the promoters of the presentdisclosure operably linked to a nucleotide sequence of interest can beused to transform any plant. In this manner, genetically modifiedplants, plant cells, plant tissue, seed, root and the like can beobtained.

As used herein, “vector” refers to a DNA molecule such as a plasmid,cosmid or bacterial phage for introducing a nucleotide construct, forexample, an expression cassette, into a host cell. Cloning vectorstypically contain one or a small number of restriction endonucleaserecognition sites at which foreign DNA sequences can be inserted in adeterminable fashion without loss of essential biological function ofthe vector, as well as a marker gene that is suitable for use in theidentification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance, hygromycin resistance or ampicillin resistance.

The methods of the disclosure involve introducing a polypeptide orpolynucleotide into a plant. As used herein, “introducing” is intendedto mean presenting to the plant the polynucleotide or polypeptide insuch a manner that the sequence gains access to the interior of a cellof the plant. The methods of the disclosure do not depend on aparticular method for introducing a sequence into a plant, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the plant. Methods for introducing polynucleotide orpolypeptides into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods andvirus-mediated methods.

A “stable transformation” is a transformation in which the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” means that a polynucleotide is introducedinto the plant and does not integrate into the genome of the plant or apolypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No.5,563,055 and Zhao, et aL, U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; 5,932,782; Tomes, et al., (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology6:923-926) and Lec1 transformation (WO 00/28058). Also see, Weissinger,et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987)Particulate Science and Technology 5:27-37 (onion); Christou, et al.,(1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et aL, (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens), all of which are herein incorporated byreference in their entirety.

In specific embodiments, the DNA constructs comprising the promotersequences of the disclosure can be provided to a plant using a varietyof transient transformation methods. Such transient transformationmethods include, but are not limited to, viral vector systems and theprecipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

In other embodiments, the polynucleotide of the disclosure may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the disclosure within a viral DNA or RNAmolecule. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta, et al.,(1996) Molecular Biotechnology 5:209-221, herein incorporated byreference in their entirety.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference intheir entirety. Briefly, the polynucleotide of the disclosure can becontained in transfer cassette flanked by two non-identicalrecombination sites. The transfer cassette is introduced into a planthaving stably incorporated into its genome a target site which isflanked by two non-identical recombination sites that correspond to thesites of the transfer cassette. An appropriate recombinase is providedand the transfer cassette is integrated at the target site. Thepolynucleotide of interest is thereby integrated at a specificchromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84, herein incorporated by reference inits entirety. These plants may then be grown, and either pollinated withthe same transformed strain or different strains and the resultingprogeny having expression of the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure thatexpression of the desired phenotypic characteristic is stably maintainedand inherited and then seeds harvested to ensure expression of thedesired phenotypic characteristic has been achieved. In this manner, thepresent disclosure provides transformed seed (also referred to as“transgenic seed”) having a nucleotide construct of the disclosure, forexample, an expression cassette of the disclosure, stably incorporatedinto its genome.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, (1988) In:Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., SanDiego, Calif., herein incorporated by reference in its entirety). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil. Preferably, theregenerated plants are self-pollinated to provide homozygous transgenicplants. Otherwise, pollen obtained from the regenerated plants iscrossed to seed-grown plants of agronomically important lines.Conversely, pollen from plants of these important lines is used topollinate regenerated plants. A transgenic plant of the embodimentscontaining a desired polynucleotide is cultivated using methods wellknown to one skilled in the art. The embodiments provide compositionsfor screening compounds that modulate expression within plants. Thevectors, cells and plants can be used for screening candidate moleculesfor agonists and antagonists of the promoters. For example, a reportergene can be operably linked to a promoter and expressed as a transgenein a plant. Compounds to be tested are added and reporter geneexpression is measured to determine the effect on promoter activity.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

The embodiments are further defined in the following Examples, in whichparts and percentages are by weight and degrees are Celsius, unlessotherwise stated. It should be understood that these Examples, whileindicating embodiments of the disclosure, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of theembodiments, and without departing from the spirit and scope thereof,can make various changes and modifications of them to adapt to varioususages and conditions. Thus, various modifications of the embodiments inaddition to those shown and described herein will be apparent to thoseskilled in the art from the foregoing description. Such modificationsare also intended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 EGS System Mutant Scheme

This approach utilizes a maternal embryo defective (embryo lethal)recessive mutation which is then maintained in an approach similar tothat used in the Sterile Inbred Maintenance System (SIMS) or SeedProduction Technology (see, U.S. Pat. Nos. 7,696,405, 7,915,398 and7,790,951). A transgenic cassette is introduced which has three parts: awild type allele to complement the embryo lethal mutation, a pollenablation PTU to prevent transgene transmission through the pollen, and aseed color marker to allow removal of a transgenic population from theseeds produced. The resultant population will be homozygous for therecessive mutant allele, but transgenically complemented. These plantsshould segregate 1:1 in the subsequent generation for viable transgenicseed and non-transgenic, non-viable, embryo-less homozygous mutants.

Schematically:

2 types of plants:

Maternal embryo defective (embryo lethal) mutant: ee

Wild type allele to complement in hemizygous state: E−

Plant is ee+E/pollen-ablation PTU/seed color marker (E is onlytransmitted through egg) When selfed: Female gametes are 50% e (embryolethal), and 50% eE (embryo viable)

-   -   Male gametes are 100% e (all pollen carrying E are ablated)

Seeds produced by these plants are 50% ee (embryo lethal)

-   -   50% eEe (normal embryo due to complementing E, colored seed)

1) Construct B, a wild-type complementing transgene/egg-cell antidoteline

2) a pollen ablation transgene

-   -   a. Multiple were demonstrated        -   i. AT-LAT52LP1 PRO:BA-BARNASE-INT        -   ii. AT-PPG1 PRO:BA-BARNASE-INT        -   iii. AT-LAT52LP2 PRO:ADP RIBOSYLASE        -   iv. AT-LAT52LP1 PRO:DMETH (Dam methylase)        -   v. AT-LAT52LP2 PRO:DMETH (Dam methylase)        -   vi. AT-PPG1 PRO:DMETH (Dam methylase)

3) a seed color marker

-   -   a. Several have been demonstrated in Arabidopsis and maize        -   i. Arabidopsis: KTI3 PRO:AC-GFP1; KTI3 PRO:AM-CYAN; RD29A            PRO:DS-RED EXPRESS; RD29A PRO:ZS-YELLOW.

4) (for self-reproducing hybrids) a parthenogenesis PTU

-   -   a. Promoters have been listed    -   b. AT-RKD2 is a CDS candidate    -   c. Promoter driving cDNA library linked to KTI3:AC-GFP1 as an        embryo reporter. This constitutes an “parthenogenesis library”    -   d. Use the Union Biometrica COPAS (Complex Object Parameter        Analyzer and Sorter) to identify GFP positive seeds        -   i. COPAS simultaneously detects optical density,            time-of-flight, RED-, Yellow-, and Green-fluorescence.        -   ii. The screen involves searching through seed for DS-RED            negative, GFP positive seeds indicating an adventitious            embryo was formed.            -   1. DS-RED negative indicates the EGS maintainer is                absent, and hence the egg cell was ablated and sexual                zygote prevented            -   2. GFP positive indicates the parthenogenesis library is                present.

Example 2 Embryogenesis Gain-of-Function Screen (EGS)

Wild type Arabidopsis plants are transformed with a constructcontaining: pollen ablation, egg cell +, and seed color marker. Plantsare then selfed to create a hemizygous transgenic population.

Hemizygous transgenic population of Arabidopsis plants are thentransformed with a construct containing egg ablation.

Seed from viable plants is grown and resultant transformed Arabidopsisplants are hemizygous for the egg ablation construct. These plants aretransformed with a construct from apomictic library containing somaticembryony and embryo color marker.

Further to describe this in more detail:

Construct A contains egg cell specific promoter:toxin gene

Construct B contains egg cell specific promoter:toxin antidote/pollenablation PTU/seed color marker

When a plant comprising both Construct A and Construct B is selfed:

Female gametes are 100% A+B (because A−only are non-viable)

Male gametes are 100% A (because A+B pollen is ablated)

Resultant seed produced is

-   -   100% (A+A)A (homozygous for construct A, hemizygous for        construct B)

Selfing this generation produces,

-   -   50% AA/B− (viable transgenic)    -   50% AA/−− (non-viable embryoless)

Resultant seeds sorted by COPAS produce approximately 50% EGS egg+ seed(viable transgenic), 50% non-fluorescent aborted seed (nonviableembryoless).

The required components are:

1) Construct A, a recessive embryo-lethal mutant/egg-cell ablation line

2) Construct B, a wild-type complementing transgene/egg-cell antidoteline

3) a pollen ablation transgene

-   -   a. Multiple were demonstrated        -   i. AT-LAT52LP1 PRO:BA-BARNASE-INT        -   ii. AT-PPG1 PRO:BA-BARNASE-INT        -   iii. AT-LAT52LP2 PRO:ADP RIBOSYLASE        -   iv. AT-LAT52LP1 PRO:DMETH (Dam methylase)        -   v. AT-LAT52LP2 PRO:DMETH (Dam methylase)        -   vi. AT-PPG1 PRO:DMETH (Dam methylase)

4) a seed color marker

-   -   a. Several have been demonstrated in Arabidopsis and maize        -   i. Arabidopsis: KTI3 PRO:AC-GFP1; KTI3 PRO:AM-CYAN; RD29A            PRO:DS-RED EXPRESS; RD29A PRO:ZS-YELLOW.

5) (for self-reproducing hybrids) a parthenogenesis PTU

-   -   a. Promoters have been listed    -   b. AT-RKD2 is a CDS candidate    -   c. Promoter driving cDNA library linked to KTI3:AC-GFP1 as an        embryo reporter. This constitutes an “parthenogenesis library”    -   d. Use the Union Biometrica COPAS (Complex Object Parameter        Analyzer and Sorter) to identify GFP positive seeds        -   i. COPAS simultaneously detects optical density,            time-of-flight, RED-, Yellow-, and Green-fluorescence.        -   ii. The screen involves searching through seed for DS-RED            negative, GFP positive seeds indicating an adventitious            embryo was formed.            -   1. DS-RED negative indicates the EGS maintainer is                absent, and hence the egg cell was ablated and sexual                zygote prevented            -   2. GFP positive indicates the parthenogenesis library is                present.

Example 4 Activity of the Expression Cassette Comprising the EggAblation Reporter AT-RKD1:Barnase-Triple Label (AT-DD45:DsRedAT-DD31:ZsYellow AT-DD65:AmCyan) in EGS Maintainer Line

FIG. 13 is a fluorescent image of a fertilized Arabidopsis embryo sacwith only remnants of the egg/zygote (red) and of the synergids (green).Mixing of the breakdown products green and red equal yellow. Centralcell appears healthy with 3-4 endosperm nuclei indicating thatfertilization did occur.

Example 5 Activity of the Expression Cassette Comprising the EggAblation Reporter AT-RKD2:Barnase-Triple Label (AT-DD45:DsRedAT-DD31:ZsYellow AT-DD65:AmCyan) in EGS Maintainer Line

FIG. 14, FIG. 15, FIG. 16, FIG. 17, FIG. 18A, FIG. 18B, FIG. 19 and FIG.20 depict several events from the same transformation construct.

FIG. 14 is a fluorescent image of a fertilized Arabidopsis embryo sacwith a zygote (red) that is in the process of breaking down, losingintegrity and appears to be “blebbing”. The persistent synergid (green)appears to be condensing and breaking down as well. Central cell appearshealthy with several endosperm nuclei indicating that fertilization didoccur.

FIG. 15 is a fluorescent image of a fertilized Arabidopsis embryo sacshowing 7-8 endosperm nuclei in a normal developing central cell. Nosign of a zygote or embryo (red) nor any sign of a synergid (green) ispresent. The endosperm may be described as developing in the absence ofan embryo.

FIG. 16 is a fluorescent image of a fertilized Arabidopsis embryo sacwith a remnant of the zygote (red) and the persistent synergid (green),where both appear to be condensing and breaking down. Central cellappears to be unhealthy and in the early stages of breaking down as isindicated by the increased vacuolation of the central cell.

FIG. 17 is a fluorescent image of 2 unfertilized Arabidopsis embryo sacsjust prior to fertilization. The embryo sac at left has a central cell(cyan) with the 2 endosperm nuclei and 2 synergids (yellow), m but islacking an egg (red). The embryo sac at right has a central cell (cyan)with the single primary endosperm nucleus, but is lacking the synergids(yellow) and the egg (red).

FIG. 18A and FIG. 18B is a fluorescent and differential interferencecontrast (DIC) fluorescent overlay imag of a fertilized Arabidopsisembryo sac. The central cell (cyan) has the single endosperm nucleusand1 synergid (yellow), but is lacking an egg (arrow).

FIG. 19 is a fluorescent image of a fertilized Arabidopsis embryo sacwith 4 endosperm nuclei in a normal developing central cell. Only a veryweak red fluorescent signal (arrow) indicative of a remnant of theembryo or zygote is present. The persistent synergid (green) is breakingdown. The endosperm is developing in the absence of an embryo.

FIG. 20 is a fluorescent image of 2 Arabidopsis embryo sacs with welldeveloped endosperm. The embryo sac at left has numerous endospermnuclei in its central cell (cyan) and at its micropylar end (arrow) is aremnant of the embryo or zygote (red). Under normal conditions thisembryo should be much more fully developed, at the heart-shaped stage.The smaller embryo sac at right has numerous endosperm nuclei (cyan) butis lacking an embryo (arrow). Synergids would have been lost by thislate stage.

What is claimed is:
 1. A method for selectively expressing gene productsin plant male tissue, comprising a) transforming a plant using anisolated nucleic acid molecule comprising a polynucleotide selected fromthe group comprising: (i) a nucleotide sequence comprising thenucleotide sequence of SEQ ID NO:53, 54, 55 and 56; (ii) a nucleotidesequence comprising a fragment or variant of the nucleotide sequence ofSEQ ID NO: 53, 54, 55 and 56, wherein the sequence initiatestranscription in a plant cell; (iii) a polynucleotide which iscomplementary to the polynucleotide of (a) or (b), b) growing the plantunder normal plant growing conditions, where the polynucleotide encodesa pollen preferred promoter which drives pollen specific expression. 2.An expression cassette comprising the polynucleotide of claim 1 operablylinked to a heterologous polynucleotide of interest.
 3. A vectorcomprising the expression cassette of claim
 2. 4. A plant cellcomprising the expression cassette of claim
 2. 5. The plant cell ofclaim 4, wherein said expression cassette is stably integrated into thegenome of the plant cell.
 6. The plant cell of claim 4, wherein saidplant cell is from a dicot.
 7. The plant cell of claim 6, wherein saiddicot is soybean.
 8. A plant comprising the expression cassette of claim2.
 9. The plant of claim 8, wherein said plant is a dicot.
 10. The plantof claim 9, wherein said dicot is soybean.
 11. The plant of claim 8,wherein said expression cassette is stably incorporated into the genomeof the plant.
 12. A transgenic seed of the plant of claim 11, whereinthe seed comprises the expression cassette.
 13. The plant of claim 8wherein the heterologous polynucleotide of interest encodes a geneproduct that is involved in cell ablation, prevention of transgenetransmission, organ development, stem cell development, cell growthstimulation, organogenesis, somatic embryogenesis initiation,self-reproducing plants and development of the apical meristem.
 14. Theplant of claim 13 wherein said gene is selected from the groupconsisting of: ADP Ribosylase, DMETH, BA-BARNASE-INT or other cellgrowth inhibitor.
 15. The plant of claim 8, wherein the heterologouspolynucleotide of interest encodes a gene product that confers droughttolerance, cold tolerance, herbicide tolerance, pathogen resistance orinsect resistance.
 16. The plant of claim 8, wherein expression of saidpolynucleotide alters the phenotype of said plant.
 17. A method forexpressing a polynucleotide in a plant or a plant cell, said methodcomprising introducing into the plant or the plant cell an expressioncassette comprising a promoter operably linked to a heterologouspolynucleotide of interest, wherein said promoter comprises a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence comprising the nucleotide sequence of SEQ ID NO:53, 54, 55 and56; (b) a nucleotide sequence comprising a fragment or variant of thenucleotide sequence of SEQ ID NO: 53, 54, 55 and 56, wherein thesequence initiates transcription in a plant cell; (c) a nucleotidesequence which is complementary to (a) or (b) where the nucleotidesequence encodes a pollen preferred promoter which drives pollenspecific expression.
 18. The method of claim 17 wherein the heterologouspolynucleotide of interest encodes a gene product that is involved incell ablation, prevention of transgene transmission organ development,stem cell development, cell growth stimulation, organogenesis, somaticembryogenesis initiation, self-reproducing plants and development of theapical meristem.
 19. The method of claim 18 wherein said gene isselected from the group consisting of: ADP Ribosylase, DMETH,BA-BARNASE-INT or other cell growth inhibitor.
 20. The method of claim17, wherein the heterologous polynucleotide of interest encodes a geneproduct that confers drought tolerance, cold tolerance, herbicidetolerance, pathogen resistance or insect resistance.
 21. The method ofclaim 17, wherein said plant is a dicot.
 22. The method of claim 21,wherein said heterologous polynucleotide of interest is expressedpreferentially in early pollen cells of said plant.
 23. A method forexpressing a polynucleotide preferentially in pollen cells of a plant,said method comprising introducing into a plant cell an expressioncassette and regenerating a plant from said plant cell, said planthaving stably incorporated into its genome the expression cassette, saidexpression cassette comprising a promoter operably linked to aheterologous polynucleotide of interest, wherein said promoter comprisesa nucleotide sequence selected from the group consisting of: (a) anucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 53,54, 55 and 56; (b) a nucleotide sequence comprising a fragment orvariant of the nucleotide sequence of SEQ ID NO: 53, 54, 55 and 56,wherein the sequence initiates transcription in a plant cell; (c) anucleotide sequence which is complementary to (a) or (b) where thepolynucleotide encodes a pollen preferred promoter which drives pollenspecific expression.
 24. The method of claim 23 wherein the heterologouspolynucleotide of interest encodes a gene product that is involved incell ablation, prevention of transgene transmission organ development,stem cell development, cell growth stimulation, organogenesis, somaticembryogenesis initiation, self-reproducing plants and development of theapical meristem.
 25. The method of claim 24 wherein said gene isselected from the group consisting of: ADP Ribosylase, DMETH,BA-BARNASE-INT or other cell growth inhibitor.
 26. The method of claim23, wherein the heterologous polynucleotide of interest encodes a geneproduct that confers drought tolerance, cold tolerance, herbicidetolerance, pathogen resistance or insect resistance.
 27. The method ofclaim 23, wherein said plant is a dicot.